Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase make up the family of pterin-dependent aromatic amino acid hydroxylases. These are nonheme iron monooxygenases which catalyze key steps in the metabolism of higher eukaryotes: catabolism of excess phenylalanine, catecholamine biosynthesis and serotonin biosynthesis, respectively. The three enzymes contain homologous catalytic domains of about 300 amino acid residues which catalyze the common chemical mechanism, activation of molecular oxygen for oxygen addition across a carbon-hydrogen bond, and discrete regulatory domains of 100-160 residues containing one or more phosphorylation sites. Mechanistic experiments are described to probe the validity of specific steps in a proposed catalytic mechanism, using both wild type tyrosine hydroxylase and several active site mutant proteins. The results of these experiments will extend our understanding of this family of enzymes and of general mechanisms of hydroxylation by biological systems. Studies of the regulatory mechanism are proposed to extend previous analyses of the effects of phosphorylation of Ser40 of tyrosine hydroxylase to the three other phosphorylation sites in order to determine if the roles of the different sites are similar or distinct. The results will enhance our understanding of the biological regulation of catecholamine biosynthesis. [unreadable] [unreadable]